Project Summary Modest protection of RV144 trial correlated with non-neutralizing antibodies targeting the V1V2 and V3 region of HIV Env and antibody-dependent cellular cytotoxicity (ADCC) activity. These findings reinvigorated the interest in HIV vaccine approaches that can induce protective Abs, neutralizing and non-neutralizing, with Fc functional competency. This proposal will investigate the role of Env signal sequence (SS) in modulating the capacity of HIV Env to elicit protective anti-Env Abs with Fc functions. The proposed study is based on our preliminary findings that, by swapping the SS of HIV isolate AA05 with SS from another isolate AC02, we produced gp120 (AA05-02SS) that induced V1V2 and V3 Ab response with increased breadth, higher titers, and most importantly greater Fc function in immunized mice. Abs induced by gp120 AA05-02SS cross-reacted with V2 peptides of JRFL, MN, HxB2 and A244, while the WT gp120 AA05 bound weakly to V2 of HxB2 only. Notably, Abs induced by AA05-02SS displayed V1V2-specific ADCP activity while the WT gp120 AA05 did not. Indeed, SS-swap altered the proportion of high-mannose and complex glycans on the AA05- 02SS vs WT AA05 gp120 as shown by mass spectrometry; these changes were at N-glycans that are in the V1V2, C2, V3 and the V4 loop of gp120. We also found that swapping AA05 and AC02 SS onto HIV REJO Env rendered REJO virus more resistant to neutralization by V1V2-specific mAbs. SS-induced changes of Env immunogenicity and virus neutralization phenotype correlated with altered Env recognition by mAbs and lectins specific for high-mannose and complex sugars. In a separate study, single mutations introduced to the Env SS of REJO or JRFL also affected oligosaccharide compositions of N-glycans on virion-associated Env, which in turn modulated Env recognition and virus sensitivity to neutralization by V1V2- and V3-specific mAbs. Hence, we propose an overall hypothesis that, by altering the Env SS, we can regulate the glycosylation of HIV Env to impact on epitope exposure/stability and immunogenicity, and by selecting a particular SS or SS residue/s we can generate Env immunogen with enhanced capacity to elicit functional Abs. We will test this idea by assessing the SS-swapped/mutant Env immunogens for changes in epitope exposure and stability by probing with Abs and for changes in N-glycan sugars by high energy C-trap dissociation mass spectrometry (Aim 1). We will immunize mice with selected SS-modified Env immunogens and compare their capacity to induce Env-specific Abs with Fc functions. We will also identify the SS signature associated with induction of functional Abs. Immunization regimen that produced Abs with or without Fc functions will be used to isolate mAbs (Aim 2). We will evaluate the protective efficacy of vaccine-induced Abs elicited by SS- modified Env immunogens in passive transfer/HIV challenge experiments using humanized mouse model (Aim 3). Data from this study will provide vital information about HIV Env SS that can be exploited to design more effective HIV vaccine capable of eliciting protective Ab response against HIV.